The present invention is directed to methods for expressing plasminogen activator in bacteria, to DNA vectors useful therein, and to protein products expressed thereby.
Blockage of blood flow in the circulatory system by the formation of blood clots is a major cause of diseases and death. The most serious problems occur when blood flow is cut off to the heart (heart attack), to the brain (stroke) or to the lungs (pulmonary embolism). To combat these problems, a group of biological compounds called the plasminogen activators functions to destroy blood clots through what is known as the fibrinolytic system. Through various mechanisms, the plasminogen activators generate the active enzyme plasmin, which in turn degrades the fibrin network of a clot to form soluble products. The two drugs which are commercially available for thrombolytic therapy which function as plasminogen activators are streptokinase, a bacterial protein, and urokinase, a serine protease isolated from human urine. Both of these drugs, however, suffer from a major drawback: they are not specific for blood clots. In addition to fibrin, they destroy such blood proteins as fibrinogen prothrombin, Factor V and Factor VIII, which may result in internal hemorrhage as a side effect of treatment. Therefore, administration of streptokinase and urokinase is usually performed by direct insertion to the site of the clot via a catheter. Since this procedure is rather intricate, it is rarely used. On the other hand, the plasminogen activators have been extracted from normal and tumor tissues and are presently classified according to the source from which they are derived: the urokinase-type plasminogen activators (uPA) and the tissue type plasminogen activators (tPA). It appears that the tPA has a higher affinity for fibrin compared to the uPA group of activators. It would thus be desirable to prepare tPA by genetic engineering techniques in order to increase the supply and variety of available tPAs.
Complementary DNA (cDNA) sequences encoding human tissue-type plasminogen activator (tPA) have been cloned and expressed in E. coli by Pennica, et al. (Nature 301: 214-221, 1983) and by Goeddel, et al. (European Patent Application Publication No. 93,619). In each of these cases, the tPA produced by the host cells was recovered through cell lysis and extraction under harsh conditions, i.e., 6M guanidine hydrochloride. The guanidine hydrochloride must subsequently be removed to permit renaturation of the protein. Goeddel, et al. show partial processing to the two-chain form of the tPA molecule, which could be inhibited by the addition of protease inhibitor.
Production of foreign proteins in bacterial cells is often hampered by difficulties in extracting the desired product from the cells. One potential solution to this problem is to direct the transformed bacterial cells to secrete the foreign protein by employing a leader peptide, but results are unpredictable. There have been attempts to produce and secrete mature foreign proteins in transformed bacterial cells using bacterial secretion signals or fused pre-sequences. Correct processing to remove the pre-sequence (bacterial or fused) has not been observed, except for limited success with some fused prepeptides. In order to obtain the desired mature foreign protein, additional (in vitro) processing is generally necessary.
It is therefore an object of the present invention to provide methods for producing mature single chain tPA in bacteria which is advantageously extracted from the cells.
It is a further object of the present invention to provide DNA vectors which are useful for expressing tPA in E. coli. 
It is yet another object of the present invention to produce in E. coli a protein having the activity of human tPA.